The aim of this proposal is to develop a novel method for the estimation of agonist activity at G protein coupled receptors. Our method involves estimating a new parameter termed the intrinsic relative activity of the agonist (IRA value), which is equivalent to the product of the observed affinity and intrinsic efficacy of an agonist expressed relative to that of a standard agonist. The only data required for estimation of IRA are the points of the concentration-response curve of the agonist. Since the IRA value is a property of the agonist-receptor- G protein complex and is probably unaffected by downstream signal transduction mechanisms, the IRA value is useful for comparing the activity of agonists across different response systems. We plan to investigate the usefulness of the IRA value for characterizing agonist activity at recombinant receptors expressed in cell lines and their native counterparts in isolated tissues. Studies are planned to estimate the IRA values of agonists in cell lines expressing M2 and M3 muscarinic and Beta 1 and Beta2 adrenoceptors and to compare these estimates with those calculated in native tissues where the former recombinant receptors are thought to mediate the responses of the tissues. We also plan to estimate both the observed affinity and relative efficacy of agonists at M3 muscarinic receptors expressed in cell lines and guinea pig ileum and to compare the product of these estimates (i.e., affinity x efficacy) with the IRA value. Some commonly used assays for investigating agonist activity are carried out on cellular homogenates where the concentration of guanine nucleotides often differs from that maintained under physiological conditions. Consequently, we plan to investigate the effects of GTP on the IRA values of agonists determined in adenylyl cyclase assays on broken cell homogenates. Our working hypothesis is that GTP reduces the observed affinity of agonists but increases their intrinsic efficacy, resulting in no change, in the IRA value of the agonist over a range of concentrations of GTP. We also plan to compare the IRA values of agonists estimated in cAMP assays on receptors linked to Gs or Gi with those measured in phosphoinositide assays in cells where the receptors are coexpressed with the promiscuous G proteins Galpha15 and Galpha16. Our working hypothesis is that the IRA values of agonists are probably, but not necessarily always, independent of the G protein with which the receptor interacts. The simple method for estimation of agonist activity described in this proposal should greatly aid in the discovery of novel therapeutic agents and in the study of the functional role of G protein coupled receptors in various physiological processes. Our proposed investigation of Galpha15 and Galpha16 is highly significant with regard to the widespread use of this G protein as a transducer for both orphan and known receptors.